The present invention relates to the production and use of transgenic animal models resulting from overexpression of galanin.
The background of U.S. Pat. No. 5,756,460 to Evans, et al. discusses galanin. Galanin is a putative neuropeptide which was first isolated from porcine small intestine in 1983. Porcine galanin is a peptide of 29 amino acid residues which was named for its N-terminal glycine and amidated C-terminal alanine residues. The cDNAs encoding galanin have been cloned from several species including: rat, porcine and bovine, revealing that galanin is a proteolytic product of a larger precursor protein known as preprogalanin. Galanin shows 90% homology between the species but little similarity to other known peptides.
Antibodies raised to porcine galanin have allowed the mapping of galanin-like-immunoreactivity (GAL-LI) to discrete regions of the central nervous system (CNS) and throughout the peripheral nervous system (PNS) of several other species, including man.
Immunohistochemical mapping of GAL-LI in the CNS has been performed most intensively in the rat where the highest concentrations have been found in the median eminance and hypothalamus. These results are consistent with more recent in situ hybridization studies where the localization of preprogalanin in the rat brain tentatively suggests the involvement of galanin in the feeding regulation of several factors ranging from water balance behaviour to blood pressure control. Similarly, radioimmunoassay of galanin in the baboon brain showed high GAL-LI in the hypothalamus and median eminance, and also GAL-LI in association with limbic structures such as the amygdala.
Immunohistochemistry and in situ studies of preprogalanin mRNA during development of the rat has shown tissue specific sex difference in galanin concentration, notably in the anterior pituitary where its expression is estrogen dependent. The overall distribution of GAL-LI and its colocalisation in discrete neuronal cells with catecholamines, scrotonin, GABA, acetylcholine and various other peptides strongly suggest a modulatory role for galanin.
A noteworthy example is the coexistence of galanin with acetylcholine in nerve fibers projecting from the basal forebrain to the hippocampus in the rat and baboon which has led to speculation that galanin may play a role in Alzheimers disease. There is, however, conflicting evidence concerning the expression of galanin in this region of the human brain.
Although the physiological role of galanin in the CNS has not yet been established its pharmacology suggests a role in neuroendocine regulation. Injection of galanin into the third ventricle of rats causes increased growth hormone and injection into the paraventricular nucleus (PVP) enhances food intake.
In the PNS, distribution of GAL-LI suggests that galanin is widespread. Galanin distribution and its pharmacology, which is diverse and often species specific, both suggest a range of physiological actions for galanin. However, some confusion may have arisen as to its pharmacological role through the use of porcine galanin in experiments involving other species.
In numerous mammalian species the highest concentrations of GAL-LI are found in the intestine, pancreas, adrenal glands, and respiratory and genitourinary tracts. Galanin action on the pancreas and its possible role in diabetes is controversial; it has been established that porcine galanin infusion in dogs, and rat and porcine galanin perfusion through the isolated rat pancreas, decrease plasma insulin levels. However, there are conflicting results concerning porcine galanin action on the pig pancreas.
In the dog, galanin also decreases somatoslatin while increasing glucagon but this may not be the case in other species.
Intravenous porcine galanin causes growth hormone secretion in a variety of species including man. However, intravenous porcine galanin infusion in man at a concentration sufficiently high to elicit an increase in growth hormone levels, does not cause the expected inhibition of insulin.
The apparent discrepancy may be due to the difference in amino acid sequence of human versus porcine galanin, or it may be simply a reflection of the species specific effects of galanin. Visualization of GAL-LI in neurons innervating the islets of several species adding to a proposal to explain the galanin induced inhibition of insulin secretion in rat B-cell lines support a neuromoclaistory role for galanin on endocrine pancreatic action. Other pharmacological effects of galanin in the PNS include the species specific stimulatory or inhibitory action of galanin on the smooth muscle activity of several mammalian species.
Galanin receptors have been identified in a hamster insulin-secreting B-cell tumor, rat and monkey brain, and smooth muscle membranes. The distribution of galanin binding correlates with that of GAL-LI and therefore supports the role of galanin in neurotransmission. It is not clear whether there are subtypes of the galanin receptor, nor which region of the peptide is responsible for binding to its receptor. Studies on the biological effect of tryptic fragments of galanin on smooth muscle preparations, in addition to auto-radiographic binding studies on Rin, 5 mf pancreatic B cell lines and on intestinal membrane preparations, present conflicting results.
The molecular biology of the galanin gene has not yet been examined in humans. Porcine preprogalanin is a 23 amino acid residue protein that comprises a signal sequence, galanin (29 amino acids) and a 59 amino acid peptide known as galanin mRNA associated peptide (GMAP). The length and structure of rat porcine and bovine preprogalanin are similar. The 20% difference in galanin amino acid homology across the species is manifest over the C-terminal end of the peptide. The sequence in all species identified to date suggests post translational cleavage of glycine extended galanin followed by amidation. GMAP is also well conserved across the species which has led to speculation that it is biologically active; it includes a region of 35 amino acids that shows 78% homology across the species and within this region a stretch of 17 residues that shows greater homology.
Galanin is widely distributed in the central and peripheral nervous system (Merchenthaler, et al., (1993); Vrontakis, et al., (1991)), and most abundantly in the hypothalamus where it may serve in the regulation of anterior pituitary hormones (Vrontakis, et al., (1991); Ottlez, et al., (1988)). In vitro studies have shown that galanin may regulate hormone secretion directly at the level of the pituitary gland (Gabriel, et al., (1988); Wynick, et al., (1993)). Central administration of galanin results in increased plasma levels of GH and PRL in a dose dependent manner (Murakami, et al., (1989); Koshiyama, et al., (1987)).
It has previously been demonstrated that galanin is dramatically upregulated by estrogen in the anterior pituitary (Vrontakis, et al., (1989); Kaplan L M et al., (1988)), primarily within the somatotrophs and lactotrophs (Steed J et al., (1989); Hyde J F, et al.). A correlation between the development of pituitary hyperplasia and the increase of galanin mRNA and peptide concentration exists, indicating that galanin might act as a mitogen in the formation of pituitary adenomas (Vrontakis M E et al, (1987); Moore J, et al., (1994); Leile V, et al., (1993)). Similarly axotomy of sensory or motor neurons dramatically increases galanin mRNA and protein (Rutherford S D, et al., (1992); Mohney R P, et al., (1994)). It has been proposed that these changes in peptide expression could be related to unknown trophic mechanisms of importance for survival and regeneration.
It would therefore be useful to have transgenic models which show the overexpresion of galanin
According to the present invention, a transgenic mammal whose somatic and germ cells having a nucleic acid construct wherein the construct includes a mammalian promoter operably linked to a cDNA genomic sequence for the overexpression of gelanin is provided. Also provided is a construct having cDNA for the overexpression of galanin. A method of making a transgenic mammal by producing a mammal having a construct for the overexpression of galanin is provided.